1. Field of the Invention
The present invention relates to the field of molecular biology and, in particular, relates to methods for the reverse transcription and amplification of ribonucleic acid (RNA) sequences.
2. Description of Related Art
The term “reverse transcriptase” describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA which can then be cloned into a vector for further manipulation.
The term “DNA polymerase” describes a class of polymerases characterized as DNA-dependent DNA polymerases. DNA polymerase show a strong discrimination against using an RNA template, as expected from their functions in vivo. Nevertheless, several laboratories have shown that some DNA polymerases are capable of in vitro reverse transcription of RNA (Karkas, 1973, Proc. Nat. Acad. Sci. USA 70:3834–3838; Gulati et al., 1974, Proc. Nat. Acad. Sci. USA 71:1035–1039; and Wittig and Wittig, 1978, Nuc. Acid Res. 5:1165–1178). Gulati et al. found that E. coli Pol I could be used to transcribe Qβ viral RNA using oligo(dT) 10 as a primer. Wittig and Wittig have shown that E. coli Pol I can be used to reverse transcribe tRNA that has been enzymatically elongated with oligo(dA). However, as Gulati et al. demonstrated, the amount of enzyme required and the small size of the cDNA product suggests that the reverse transcriptase activity of E. coli Pol I has little practical value.
T. aquaticus (Taq) DNA polymerase, a thermostable DNA polymerase, has been reported to inefficiently synthesize cDNA using Mg+2 as the divalent metal ion (Jones and Foulkes, 1989, Nuc. Acids. Res. 176:8387–8388). Tse and Forget, 1990, Gene 88:293–296; and Shaffer et al., 1990, Anal. Biochem. 190:292–296, have described methods for amplifying RNA using Taq DNA polymerase and Mg+2 ion. However, the methods are inefficient and insensitive.
Amplification of nucleic acid sequences, both RNA and DNA, is described in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188; each incorporated herein by reference. A preferred method, the polymerase chain reaction (PCR), typically is carried out using a thermostable DNA polymerase, such as Taq DNA polymerase, which is able to withstand the temperatures used to denature the amplified product in each cycle. PCR is now well known in the art and has been described extensively in the scientific literature. See, for example, PCR Applications, 1999, (Innis et al., eds., Academic Press, San Diego), PCR Strategies, 1995, (Innis et al., eds., Academic Press, San Diego); PCR Protocols, 1990, (Innis et al., eds., Academic Press, San Diego); and PCR Technology, 1989, (Erlich, ed., Stockton Press, New York); each incorporated herein by reference. Commercial vendors, such as PE Biosystems (Foster City, Calif.) market PCR reagents and publish PCR protocols. A review of amplification methods is provided in Abramson and Myers, 1993, Current Opinion in Biotechnology 4:41–47, incorporated herein by reference.
Because reverse transcription using Taq DNA polymerase in a magnesium ion buffer was too inefficient to be practical, PCR amplification starting with an RNA template initially was carried out by first reverse-transcribing the target RNA using, for example, a non-thermostable viral reverse transcriptase such as Molony Murine Leukemia Virus Reverse Transcriptase (MoMuLV RT) or AMV-RT, and then amplifying the resulting cDNA using a thermostable DNA polymerase.
A significant advance was achieved with the discovery that a thermostable DNA polymerase could be used to efficiently reverse transcribe an RNA template by carrying out the reaction in a manganese buffer (Mn+2), rather than a magnesium (Mg+2) buffer, as is preferred for primer extension using a DNA template. Efficient Mn+2-activated reverse transcription using a thermostable DNA polymerase is described in U.S. Pat. Nos. 5,310,652; 5,322,770; 5,407,800; 5,641,864; 5,561,058; and 5,693,517, all incorporated herein by reference. As both the synthesis of cDNA from an RNA template and the synthesis of DNA from a DNA template can be carried out in a Mn+2 buffer, the use of a Mn+2 buffer enables single-enzyme, coupled reverse transcription/amplification reactions (see also Myers and Sigua, 1995, in PCR Strategies, supra, chapter 5).